Concentration measuring method, concentration test instrument, and concentration measuring apparatus

ABSTRACT

The present invention relates to technology for constructing a reaction system including a test target, an oxidation-reduction enzyme, and an electron mediator, and measuring the concentration of the test target by an electrochemical process. A Ru compound is used as the electron mediator. The present invention provides a concentration test instrument including a substrate, first and second electrodes formed on the substrate, and a reagent layer formed as a solid. The reagent layer contains an oxidation-reduction enzyme and a Ru compound, and is constituted so as to dissolve and construct a liquid phase reaction system when a sample liquid is supplied.

This application is a 371 National Stage Entry of PCT/JP02/08855 filedon Aug. 30, 2002.

TECHNICAL FIELD

The present invention relates to technology for measuring aconcentration of a test target (such as glucose or cholesterol)contained in a sample liquid (such as blood or another such biologicalsample, or a prepared liquid thereof).

BACKGROUND ART

Enzyme reactions are used as a way to quantify glucose concentration. Ina typical case, glucose oxidase (GOD) is used as the enzyme. GOD is anenzyme which is linked to flavin adenine dinucleotide (FAD), which is acoenzyme. The enzyme reaction of glucose when GOD is used proceedsaccording to the following chemical formula (In the formula, FADH₂ isthe reduction type of the FAD).Glucose+GOD/FAD→δ-Gluconolactone+GOD/FADH₂

When blood sugar levels are measured in a clinical setting, glucoseconcentrations are sometimes quantified by measuring the change inabsorbance, which corresponds to the change in glucose concentration.However, the most common method is to measure the glucose concentrationby amperometry. Amperometry is widely employed as a method for measuringglucose concentration in portable blood sugar measurement devices.

An example of how blood sugar is measured by amperometry is given below,for the case of measuring oxidation current. In the first step, areaction system is constructed using blood, an enzyme, and an oxidativeelectron transfer medium (mediator). The result is that theabove-mentioned enzyme reaction proceeds while a reductive mediator isproduced by an oxidation-reduction reaction between the mediator and theFADH₂ produced by this enzyme reaction. Potassium ferricyanide iscommonly used as a mediator, in which case the reaction can be expressedby the following chemical formula.GOD/FADH₂+2[Fe(CN)₆]³⁻→GOD/FAD+2[Fe(CN)₆]⁴⁻+2H⁺

Next, in the second step, voltage is applied to the reaction systemusing a pair of electrodes, which oxidizes the potassium ferrocyanide(releases electrons) and produces potassium ferricyanide as shown in thefollowing chemical formula. The electrodes originating in the potassiumferrocyanide are supplied to the anode.[Fe(CN)₆]⁴⁻→[Fe(CN)₆]³⁻+e⁻

In the third step, the oxidation current value attributable to voltageapplication is measured, and the glucose concentration is computed onthe basis of this measured value.

When blood sugar is measured using a portable blood sugar measurementdevice, a glucose sensor is used in which a reagent layer containing anenzyme and a mediator is formed between electrodes, and a reactionsystem is constituted between the electrodes by supplying blood to thereagent layer. This glucose sensor is installed in a portable bloodsugar device, voltage is applied between the electrodes, the oxidationcurrent value is measured, and the glucose concentration in the blood isquantified on the basis of this oxidation current value.

As discussed above, GOD is usually used as the enzyme, and potassiumferricyanide as the mediator. Nevertheless, in a reaction systemcombining GOD with potassium ferricyanide, the problems discussed beloware encountered with a method for measuring glucose concentration by anelectrochemical process, typified by amperometry.

The first of these problems is the effect of reductive substances. Forinstance, if we consider the measurement of glucose concentration inblood, there are reductive substances (such as ascorbic acid,glutathione, and Fe(II)²⁺) coexisted in the blood in addition toglucose. If a reductive substance other than potassium cyanide ispresent when voltage is applied to the reaction system, electronsoriginating in the oxidation of the reductive substance caused byvoltage application will be supplied to the electrodes in addition tothe electrons originating in the potassium ferrocyanide. As a result,the measured current value will include background current (noise)attributable to the electron transfer of the reductive substance.Accordingly, the measured glucose concentration will end up beinggreater than the actual glucose concentration. The greater is the amountof voltage applied between the electrodes, the more types and quantityof reductive substances that are oxidized, and the more pronounced isthis measurement error. Therefore, when potassium ferricyanide is usedas the mediator, blood sugar cannot be measured accurately unless thefinal concentration is determined by correcting the measured value. Thiseffect of reductive substances is not limited to when blood sugar ismeasured, and is similarly encountered with other components when theconcentration is computed on the basis of the oxidation current value.

Another problem pertains to the storage stability of the glucose sensorwhen glucose concentration is measured with a portable blood sugarmeasurement device and a glucose sensor. Potassium ferricyanide issusceptible to the effects of light and water, and when exposed tothese, receives electrons from sources other than glucose and turns intoreductive potassium ferrocyanide. If this happens, then the reactionsystem will contain both potassium ferrocyanide that has been renderedreductive by enzyme reaction, and potassium ferrocyanide that has beenrendered reductive by exposure. As a result, just as with the reductivesubstance problem described above, the oxidation current during voltageapplication includes background current originating in the potassiumferrocyanide resulting from exposure. Consequently, the measured glucoseconcentration ends up being greater than the actual glucoseconcentration. To minimize this problem, the glucose sensor must besealed in a pouch made from a light-blocking material, for example, sothat the reagent layer in the glucose sensor is not exposed.Furthermore, to extend the service life of the glucose sensor, it has tobe sealed in a moisture-tight state by performing nitrogen replacementor other such treatment in order to avoid exposure to moisture, and thiscomplicates manufacture and drives up the cost when the glucose sensoris mass-produced on an industrial scale.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide technology thatreduces the effect of background current at low cost, and allows theconcentration of a test target in a sample liquid to be measured moreaccurately.

According to a first aspect of the present invention, there is provideda method for measuring the concentration of a test target, whereby areaction system is constituted to include the test target, anoxidation-reduction enzyme and an electron mediator, and then theconcentration of the test target is measured by utilizing anelectrochemical process. For the electron mediator, use is made of a Rucompound.

The above concentration measuring method may preferably comprise a firststep of producing a reductant of the Ru compound in the reaction system,a second step of applying voltage to the reaction system to oxidize thereductant, and measuring the response current value correlated with thequantity of electrons released by the reductant at this time, and athird step of calculating the concentration of the test target on thebasis of the response current value measured in the second step.

With the method of the present invention, the first step may beconducted with the reaction system in a voltage non-application stateand the second step then conducted with the reaction system in a voltageapplication state, or the first and second steps may be conductedsimultaneously with the reaction system in a voltage application statecontinuously from the time the sample liquid containing at least thetest target is supplied.

The voltage applied between the first and second electrodes in thesecond step is preferably a constant potential, and the value thereof ispreferably at least a standard oxidation-reduction potential (versus astandard hydrogen electrode) between the reductive Ru(II) complex andthe oxidative Ru(III) complex, and less than a standardoxidation-reduction potential (versus a standard hydrogen electrode)between ferrocyanide ions and ferricyanide ions. The constant voltageapplied between the first and second electrodes may be 100 to 500 mV,for example, and more preferably 100 to 300 mV.

Preferably, the first step lasts from 0 to 10 seconds, and the currentvalue measured after a specific amount of time has elapsed (at least 3seconds) from the start of the second step is employed as acomputational current value that serves as the basis for computation ofthe glucose concentration in the third step. Even more preferably, thefirst step lasts from 0 to 3 seconds, and the current value measuredafter a specific amount of time has elapsed (3 to 5 seconds) from thestart of the second step is employed as the computational current value.

According to a second aspect of the present invention, there is provideda concentration test instrument comprising a substrate, first and secondelectrodes formed on the substrate, and a reagent layer formed as asolid. The reagent layer may comprise an oxidation-reduction enzyme anda Ru compound, and may be constituted so as to dissolve and construct aliquid phase reaction system when a sample liquid containing the testtarget is supplied.

Preferably, the reagent layer is constituted such that when the sampleliquid is supplied, an oxidation-reduction enzyme and a Ru compound areboth present in the liquid phase reaction system.

Preferably, in the first and second aspects of the present invention,the Ru compound is present in the reaction system as an oxidative Rucomplex. There are no particular restrictions on the type of ligand inthe Ru complex as long as the complex functions as a mediator (electrontransfer medium), but it is preferable to use an oxidative typeexpressed by the following chemical formula.[Ru(NH₃)₅X]^(n+)

Examples of X in the chemical formula include NH₃, a halogen ion, CN,pyridine, nicotinamide, and H₂O, but NH₃ or a halogen ion is preferable.n⁺ in the chemical formula is the valence of the oxidative Ru(III)complex as determined by the type of X.

If the Ru compound is an oxidative Ru(III) complex, then the electrontransfer system is selected so that the reductive Ru(II) complex will beproduced by only two reactions: an oxidation reaction of the measurementtest target catalyzed by the oxidation-reduction enzyme, and a reductionreaction of the oxidative Ru(III) complex.

The reaction system is constituted, for example, as a uniform orsubstantially uniform liquid phase reaction system in which a relativelysmall amount of the oxidation-reduction enzyme is dispersed uniformly orsubstantially uniformly with respect to a relatively large amount of theoxidative Ru(III) complex. In this case, the reductant is producedsubstantially uniformly in every location of the reaction system.

Examples of the test target include glucose, cholesterol, lactic acid,and ascorbic acid.

The oxidation-reduction enzyme is selected according to the type of testtarget, but preferably is at least one type selected from the groupconsisting of glucose dehydrogenase (GDH) (including the αGDH and CyGDHdiscussed below), glucose oxidase (GOD), cholesterol dehydrogenase,cholesterol oxidase, lactic acid dehydrogenase, lactic acid oxidase,ascorbic acid dehydrogenase, ascorbic acid oxidase, alcoholdehydrogenase, alcohol oxidase, fructose dehydrogenase, 3-hydroxybutyricacid dehydrogenase, pyruvic acid oxidase, NADH oxidase, uric acidoxidase (uricase), urease, and dihydrolipoamide dehydrogenase(diaphorase).

With the present invention, examples of GDH that can be used includetypes in which pyrroquinoline quinone (PQQ), nicotinamide adeninedinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP),or another such compound serves as a coenzyme, as well as αGDH, CyGDH,and so forth. It is preferable for the GDH to be αGDH, CyGDH, or acompound in which PQQ serves as a coenzyme (PQQGDH).

αGDH contains a GDH-active protein whose molecular weight isapproximately 60 kDa in SDS-polyacrylamide gel electrophoresis underreduction conditions as subunits having glucose dehydrogenationactivity. CyGDH, meanwhile, contains as subunits the above-mentionedGDH-active protein and an electron mediator protein (cytochrome C) whosemolecular weight in SDS-polyacrylamide gel electrophoresis underreduction conditions is approximately 43 kDa. The GDH can also be onefurther having subunits other than a GDH-active protein and cytochromeC.

CyGDH can be obtained by refining an enzyme externally secreted by amicrobe belonging to Burkholderia cepacia, or by refining an enzymefound internally in this microbe. αGDH, meanwhile, can be obtained byforming a transformant implanted with a gene coding for the expressionof αGDH collected from a microbe belonging to Burkholderia cepacia, forexample, and refining an enzyme externally secreted from thistransformant, or refining an enzyme found internally in thistransformant.

As for the microbe belonging to Burkholderia cepacia, for example,Burkholderia cepacia KS1 strain can be used. This KS1 strain depositedon Sep. 25, 2000 as microorganism deposit number FERM BP-7306 with thePatent Organism Depositary of the National Institute of AdvancedIndustrial Science and Technology (Chuo No. 6, 1-1, Higashi 1-chome,Tsukuba-shi, Ibaraki, Japan, 305-8566).

According to a third aspect of the present invention, there is provideda concentration measuring apparatus which is used together with aconcentration test instrument including a reagent layer a firstelectrode and a second electrode, where the reagent layer contains a Rucompound as an oxidation-reduction enzyme. The measuring apparatusincludes a voltage applier for applying voltage between the first andsecond electrodes, a current value measurer for measuring the responsecurrent value when voltage has been applied between the first and secondelectrodes, and a computer for computing the concentration of the testtarget on the basis of the response current value.

Preferably, the concentration measuring apparatus may further comprise acontroller for controlling the voltage application performed by thevoltage applier, or for controlling the current value measurementperformed by the current value measurer.

The above controller is constituted, for example, such that the voltageapplied by the voltage applier is controlled to be a constant voltageselected from a range of 100 to 500 mV, and preferably 100 to 300 mV.The controller may also be constituted such that the voltage applied bythe voltage applier is controlled to be a constant voltage selected froma range of at least a standard oxidation-reduction potential (versus astandard hydrogen electrode) between the oxidant and reductant of the Rucompound, and less than a standard oxidation-reduction potential (versusa standard hydrogen electrode) between ferrocyanide ions andferricyanide ions.

Preferably, the concentration measuring apparatus of the presentinvention may further comprise a detector for detecting that a sampleliquid has been supplied to the reagent layer of the concentration testinstrument. The controller is constituted, for example, so as to controlthe voltage applier such that no voltage is applied between the firstand second electrodes during a first specific period of 0 to 10 secondsafter the detector has detected that a sample liquid has been suppliedto the reagent layer. In this case, the control means controls thevoltage applier such that a specific potential is applied between thefirst and second electrodes by the voltage applier starting at the pointwhen the first time period has elapsed. The control means is furtherconstituted such that the response current value used for concentrationcomputation by the computer is measured by the current value measurer ata point when a second specific time period of at least 3 seconds haselapsed after the start of the previous application of the specificpotential. Preferably, the first specific time period is 0 to 3 seconds,and the second specific time period is 3 to 5 seconds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the basic structure in aglucose concentration measuring apparatus according to the presentinvention;

FIG. 2 is an overall oblique perspective view illustrating a glucosesensor used in the glucose concentration measuring apparatus in FIG. 1;

FIG. 3 is an exploded oblique perspective view of the glucose sensor inFIG. 2;

FIG. 4A is a diagram of the electron transfer system in a reactionsystem including PQQ-GDH and a Ru complex, and FIG. 4B is a diagram ofthe electron transfer system in a reaction system including GOD and a Rucomplex;

FIG. 5 is a graph of the change over time in the voltage applied to thefirst and second electrodes and the response current value inmeasurement of the glucose concentration;

FIG. 6 is a graph of the CV waveforms of a glucose sensor 1 of thepresent invention and a comparative glucose sensor;

FIG. 7 is a graph of the effect of applied voltage value;

FIG. 8 is a graph of the change over time in the response current whenvoltage is applied to the reagent layer (closed circuit) after thecircuit has been open for a specific time period after the supply ofwhole blood to a reagent layer in which a Ru complex is used;

FIG. 9 is a graph of the change over time in the response current whenvoltage is applied to the reagent layer (closed circuit) after thecircuit has been open for a specific time period after the supply ofwhole blood to a reagent layer in which an Fe complex is used;

FIG. 10 is a graph of the response current value 5 seconds after thestart of voltage application when a voltage of 500 mV is applied 10seconds after whole blood is supplied to the reagent layer, for severaltypes of whole blood with different glucose concentrations;

FIG. 11 is a graph of the response current value 5 seconds after thestart of voltage application when a voltage of 250 mV is applied 10seconds after whole blood is supplied to the reagent layer, for severaltypes of whole blood with different glucose concentrations;

FIG. 12 is a bar graph of the response current value (backgroundcurrent) for whole blood with a glucose concentration of 0 in the graphsshown in FIGS. 10 and 11, given separately for an Fe complex and a Rucomplex;

FIG. 13 is a graph of an evaluation of the effect of exposure tomoisture from the response current value when a standard solution issupplied to the reagent layer;

FIG. 14 is a graph of an evaluation of the dispersibility of a Rucomplex from the response current value when a standard solution issupplied to the reagent layer;

FIG. 15 is a graph of the correlation between glucose concentration andresponse current value for glucose sensors having different reagentlayer formulations (oxidation-reduction enzymes); and

FIG. 16 is a graph of the correlation between glucose concentration andresponse current value for glucose sensors in which GOD is used as theoxidation-reduction enzyme.

BEST MODE FOR CARRYING OUT THE INVENTION

Preferred embodiments of the present invention will now be describedwith reference to the drawings. In these embodiments, the descriptionwill be for examples of a glucose concentration measuring apparatus andglucose sensor constituted such that the glucose concentration in asample liquid is measured. However, the present invention is not limitedto the measurement of glucose concentration, and can also be applied tothe measurement of other components.

As shown in FIG. 1, a glucose concentration measuring apparatus 1 uses aglucose sensor 2 to measure the glucose concentration in a glucosesolution such as blood. This glucose concentration measuring apparatus 1comprises a voltage applier 3, a current measurer 4, a detector 5, acontroller 6, a computer 7, and a display unit 8.

As clearly shown in FIGS. 2 and 3, the glucose sensor 2 includes a coverplate 20, a spacer 21, and a base plate 22. A channel 25 is created bythese members.

A hole 23 is made in the cover plate 20, and a slit 24 that communicateswith the hole 23 and is open at its distal end 24a is provided to thespacer 21. The channel 25 communicates with the outside via the hole 23and the open distal end 24a of the slit 24. The distal end 24aconstitutes a sample liquid introduction opening 25a. Glucose solutionsupplied through this sample liquid introduction opening 25a moves bycapillary action through the channel 25 toward the hole 23.

A first electrode 26, a second electrode 27, and a reagent layer 28 areprovided on the upper surface 22a of the base plate 22.

The first and second electrodes 26 and 27 generally extend in thelongitudinal direction of the base 22. The first and second electrodes26 and 27 have at their ends 26A and 27A a working portion 26a and acounterpart portion 27a extending in parallel to the shorter sides ofthe base plate 22.

The upper surface 22a of the base plate 22 is covered by an insulatingfilm 29 so as to expose the working portion 26a of the first electrode26, the counterpart portion 27a of the second electrode 27, and theopposite ends 26b and 27b of the first and second electrodes 26 and 27.As discussed below, the opposite ends 26b and 27b of the first andsecond electrodes 26 and 27 constitute terminals for providing contactwith first and second contacts 3a and 3b (see FIG. 1) of the glucoseconcentration measuring apparatus 1.

The reagent layer 28 is, for example, in solid form and provided so asto span the distance between the working portion 26a and the counterpartportion 27a. This reagent layer 28 includes, for example, a relativelylarge amount of mediator (electron transfer medium) and a relativelysmall amount of the oxidation-reduction enzyme. The reagent layer 28 isformed, for example, by applying a coating of paint, in which themediator and the oxidation-reduction enzyme are substantially uniformlydispersed, so as to span the distance between the first and secondelectrodes 26 and 27, and then drying this coating. When the reagentlayer 28 is formed in this way, it becomes a single, solid layer inwhich the oxidation-reduction enzyme is substantially uniformlydispersed in the mediator, and is readily dissolved by the supply of theglucose solution.

It is preferable to use glucose dehydrogenase (GDH) or glucose oxidase(GOD) as the oxidation-reduction enzyme. The GDH can be a type in whichsuch compounds as pyrroquinoline quinone (PQQ), nicotinamide adeninedinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP)serve as a coenzyme, or can be αGDH or CyGDH. Of these GDHs, it ispreferable to use αGDH, CyGDH, or a compound in which PQQ serves as acoenzyme (i.e., PQQGDH).

A Ru complex, for example, is used as the mediator. There are noparticular restrictions on the type of ligand in the Ru complex as longas the complex functions as an electron transfer medium, but it ispreferable to use an oxidative type expressed by the following chemicalformula.[Ru(NH₃)₅X]^(n+)

Examples of X in the chemical formula include NH₃, a halogen ion, CN,pyridine, nicotinamide, and H₂O, but NH₃ or a halogen ion is preferable.n⁺ in the chemical formula is the valence of the oxidative Ru(III)complex, which is determined by the type of X.

Ru complexes are usually in the form of an oxidative type (III) becausereductive types (II) are unstable. Accordingly, the Ru complex will notreadily undergo undesirable reduction even when exposed to light orwater when mixed into the reagent layer 28 of the glucose sensor 2.Another characteristic of a Ru complex is that it does not readilycrystallize and can be suitably maintained in the form of a micropowder.Another advantage, at least for combinations of Ru complex and PQQGDH,is fast electron transfer.

The voltage applier 3 shown in FIG. 1 applies a constant voltage betweenthe terminal 26b of the first electrode 26 and the terminal 27b of thesecond electrode 27. The voltage applier 3 is designed so that when theglucose sensor 2 is mounted in its mounting component (not shown)provided to the glucose concentration measuring apparatus 1, there iselectrical continuity between the terminals 26b and 27b of the glucosesensor 2 via the first and second contacts 3a and 3b. A DC power supplysuch as a dry cell or a rechargeable cell is used as the voltage applier3.

The current measurer 4 measures the response current value correlatedwith the quantity of electrons released from the reductive Ru(II)complex of the reagent layer 28 when voltage is applied between thefirst and second electrodes 26 and 27.

After the glucose sensor 2 is mounted in the glucose concentrationmeasuring apparatus 1, the detector 5 detects whether or not a glucosesolution has been supplied to the reagent layer 28 and measurement ofthe glucose concentration is possible.

The controller 6 controls the voltage applier 3 and selects betweenstates in which voltage is applied (closed circuit) and is not applied(open circuit) between the first and second electrodes 26 and 27. Thecontroller 6 also controls the current value measurement timing in thecurrent measurer 4.

The computer 7 computes the glucose concentration in the glucosesolution according to the response current value measured by the currentmeasurer 4.

The detector 5, the controller 6, and the computer 7 are eachconstituted by a CPU and a memory such as a ROM or RAM, for example, butit is also possible to constitute all of the detector 5, the controller6, and the computer 7 by connecting a plurality of memories to a singleCPU. The computation results of the computer 7 are displayed by thedisplay unit 8. The display unit 8 is constituted by an LCD or the like.

Next, the procedure for measuring the glucose concentration in a glucosesolution will be described through reference to FIGS. 4 and 5 inaddition to FIGS. 1 to 3.

As is clearly shown in FIG. 1, first the glucose sensor 2 is installedin the glucose concentration measuring apparatus 1. As a result, theterminals 26b and 27b of the first and second electrodes 26 and 27 ofthe glucose sensor 2 come into contact with the first and secondcontacts 3a and 3b of the glucose measuring apparatus 1. As wasmentioned above, there is electrical continuity between the first andsecond electrodes 26 and 27 and the voltage applier 3 in this state. Inactual measurement, a constant voltage is applied between the first andsecond electrodes 26 and 27 by the voltage applier 3 under the controlof the controller 6 even before the glucose solution is supplied to theglucose sensor 2.

The constant voltage applied between the first and second electrodes 26and 27 is set to within a range of 100 to 500 mV, for instance.Preferably, the constant voltage is at least a standardoxidation-reduction potential (versus a standard hydrogen electrode)between the reductive Ru(II) complex and the oxidative Ru(III) complex,and less than a standard oxidation-reduction potential (versus astandard hydrogen electrode) between ferrocyanide ions and ferricyanideions. The standard oxidation-reduction potential of a Ru complex variessomewhat with the type of ligands, but is roughly +100 mV, while that offerricyanide ions is +360 mV. Therefore, the constant voltage appliedbetween the first and second electrodes 26 and 27 by the voltage applier3 is selected from a range of 100 to 350 mV, for example. It wasdiscussed above that it is best for the Ru complex to be an oxidativetype expressed by [Ru(NH₃)₈]³⁺ (or a reductive type expressed by[Ru(NH₃)₆]²⁺). Here again, the constant voltage is preferably 100 to 350mV, and even more preferably 100 to 300 mV.

Next, a glucose solution such as blood is supplied through the sampleliquid introduction opening 25a of the glucose sensor 2. The glucosesolution moves by capillary action through the channel 25 of the glucosesensor 2. In the course of this movement the glucose solution dissolvesthe reagent layer 28.

As touched upon above, since a Ru complex does not readily crystallizeand can be suitably maintained in the form of a micropowder, if a Rucomplex is contained in the form of a micropowder in the reagent layer28, the entire reagent layer 28 will readily and instantly dissolve whenthe glucose solution is supplied. Because the reagent layer 28 comprisesa Ru complex dispersed in an oxidation-reduction enzyme, an enzymereaction occurs uniformly at every location of the reagent layer 28,which allows the glucose concentration to be measured accurately in ashort time.

Meanwhile, if a glucose solution is supplied to the reagent layer 28,the glucose is oxidized into gluconolactone and the mediator is madeinto a reductive type by the oxidation-reduction enzyme. Since themediator is substantially uniformly dispersed in the reagent layer 28, areductive mediator is produced spontaneously, without any voltage beingapplied, substantially uniformly at every location of the reagent layer28. The gluconolactone becomes gluconic acid without the help of theenzyme.

Here, FIG. 4A is a diagram of the electron transfer system when[Ru(III)(NH₃)₆]³⁺ is used as the mediator and PQQGDH is used as theoxidation-reduction enzyme, while FIG. 4B is a diagram of the electrontransfer system when [Ru(III)(NH₃)₆]³⁺ is used as the mediator and GODis used as the oxidation-reduction enzyme.

In the example depicted in FIGS. 4A and 4B, in a state in which constantvoltage is applied between the first and second electrodes 26 and 27 viathe two terminals 26b and 27b, the reductive Ru(II) complex present inthe reagent layer 28 moves to the working portion 26a side of the firstelectrode 26, releases electrons to this working portion 26a, andcreates an oxidative Ru(II) complex. Therefore, in a state in whichconstant voltage is applied between the first and second electrodes 26and 27 by the voltage applier 3, the quantity of electrons given off bythe reductive Ru(II) complex is measured as the response current valueby the current measurer 4 via the first electrode 26 and the firstcontact 3a. This response current value is correlated with the quantityof electrons originating in the reductive Ru(II) complex that has movedthrough the reagent layer 28 as a result of voltage application, and isknown as the diffusion current.

Meanwhile, the response current value measured by the current measurer 4is monitored by the detector 5, and as shown in FIG. 5, the detector 5detects that the glucose solution has been supplied to the reagent layer28 and the reagent layer 28 has dissolved at the point t₀ when theresponse current value exceeds a threshold I₁ (such as 2 to 3 μA).

When the detector 5 has detected that the glucose solution has beensupplied, the controller 6 controls the voltage applier 3 and halts theapplication of voltage between the first and second electrodes 26 and27. Since the reductive Ru(II) complex is not oxidized while voltageapplication is halted, the reductive Ru(II) complex accumulates as aresult of the glucose oxidation reaction and the mediator reductionreaction brought about by the oxidation-reduction enzyme. At a point t₁when a specific amount of time has elapsed (such as t₁-t₀=0 to 10seconds, and preferably 0 to 3 seconds), a constant voltage V is appliedbetween the first and second electrodes 26 and 27 by the voltage applier3 under the control of the controller 6. Even after the detector 5 hasdetected that the glucose solution has been supplied, application ofvoltage may be continued so that the produced reductive Ru(II) complexis successively moved to the working portion 26a and the diffusioncurrent is measured.

Here, as shown in FIGS. 4A and 4B, a reductive Ru(II) complex releaseselectrons e⁻ to become an oxidative Ru(III) complex. When otherreductive substances are present in the glucose solution along with areductive Ru(II) complex, these substances also release electrons inaccording to the type and amount of component corresponding to theapplied voltage, and become oxidative.

The electrons released by the reductive Ru(II) complex and any otherreductive substances are supplied to the working portion 26a of thefirst electrode 26 and are measured as the response current value by thecurrent measurer 4 via the first contact 3a. Therefore, the responsecurrent value that is actually measured includes that produced byelectrons originating in the coexistent substances that became oxidativeupon the application of voltage. The probability (proportion) at whichthe coexistent substances that were reductive release electrons andbecome oxidative is dependent on the amount of voltage applied to thefirst and second electrodes 26 and 27; the more voltage applied, themore types of the coexistent substances release electrons and thegreater the total amount of electrons released by the individualsubstances. Also, the reductive R(II) complex can include not only onethat has been given electrons in the oxidation-reduction reaction withthe oxidation-reduction enzyme, but also one that has been made intoreductive Ru(II) through exposure to water or light. Accordingly, theresponse current value that is actually measured can include backgroundcurrent attributable to the reductive Ru(II) originating in electrodesfrom something other than an enzyme reaction, or background noise due tothe coexistent substance that are present.

In contrast, according to the present embodiment, as is clear from FIG.5, the constant voltage V applied to the first and second electrodes 26and 27 is the same as the constant voltage V applied up to the pointwhen the detector detects that the glucose solution has been supplied tothe reagent layer 28. Specifically, the reapplied constant voltage V isbetween 100 and 350 mV, and preferably 100 to 300 mV, which is less thanthe standard oxidation-reduction potential of a ferricyanide ion. Withthis, the voltage applied to the first and second electrodes 26 and 27is less than when a ferricyanide ion (potassium) is used as themediator. This makes it possible to suppress the oxidation (release ofelectrons) of the reductive coexistent substances such as ascorbic acidor glutathione that are also present when blood or the like is used asthe glucose solution, which would otherwise occur upon the applicationof voltage. This reduces the background current caused by the effect ofthe reductive coexistent substances present in the solution. As aresult, it is possible to compute the concentration with good precisioneven without factoring in the effect of these reductive coexistentsubstances and correcting the measured values.

Also, because an oxidative Ru(III) complex is far more stable than areductive Ru(II) complex, this Ru complex is less apt to decompose inthe presence of moisture or under optical irradiation, and most of itremains as oxidative Ru(III) until given electrons by an enzymereaction. Therefore, the proportion of Ru(III) complex that has beenrendered reductive by electrons from sources other than an enzymereaction is far smaller, and this again allows background current to bereduced. Accordingly, there is no need to give much thought to theeffect of moisture in the storage of the glucose sensor 2, so there isno need to reduce the amount of moisture by means of nitrogenreplacement or the like. As a result, manufacturing is correspondinglyeasier when the glucose sensor 2 is mass-produced on an industrialscale, and this keeps the cost lower.

Furthermore, in the present embodiment, diffusion current based on thereductive Ru(II) complex produced by the entire reagent layer 28 ismeasured as the response current. In other words, since the twooxidation-reduction reactions shown in FIGS. 4A and 4B occur at everylocation of the reagent layer 28, the glucose reaction is concluded assoon as the glucose solution is supplied. Accordingly, if the glucoseconcentration is about 600 mg/dL, the oxidative Ru(III) complex will beconverted into reductive Ru(II) in an amount corresponding to theglucose concentration at the point when the response current value ismeasured, such as 5 seconds after the glucose is supplied. Therefore,the response current value will be relatively large (on the μA level)even through the glucose concentration is on the 100 mg/dL level, and istherefore less affected by noise from electromagnetic waves and soforth. This means that the glucose concentration can be measured withgood precision without having to take such steps as ensuring a largeelectrode surface area. Also, high concentration levels are difficult tomeasure when, for example, a mediator or enzyme is fixed to electrodes,the enzyme is subjected to a catalytic reaction on just the surface ofthe electrodes, electrons are exchanged between the mediator and theelectrodes, and the amount of electron movement here (catalyst current)is measured. In other words, if even one of the plurality of reactionsparticipating in the exchange of electrons between glucose and theelectrodes is slower than the other reactions, that reaction will becomethe rate-limiting stage, and even if glucose is supplied over a specificconcentration, the response current value will not rise over a specificvalue. Consequently, the response current value gradually approaches aconstant value within a range in which the glucose concentration isrelative high, making it difficult to measure high concentrations. Incontrast, when diffusion current value is measured, the response currentvalue is measured at the point when the glucose reaction has actuallyconcluded, so glucose concentrations can be appropriately measured evenat relatively high concentration levels.

Meanwhile, the computer 7 computes the glucose concentration in aglucose solution on the basis of the response current I₂ measured by thecurrent measurer 4 at the point t₂ when a specific time (such ast₂-t₁=at least 3 seconds, and preferably 3 to 5 seconds) has elapsedsince the reapplication of voltage between the first and secondelectrodes 26 and 27. The glucose concentration is computed byconverting the response current value into a voltage value, thenchecking this voltage value against a calibration curve which isproduced ahead of time and expresses the relationship between voltageand glucose concentration. This calibration curve is, for example,converted into data and stored in a ROM along with the program forexecuting computation. The glucose concentration is computed byutilizing a CPU or RAM to execute the program stored in this ROM.

EXAMPLES

It will be proven below by Examples 1 to 8 that when a Ru complex isused as a mediator in the measurement of glucose concentration byutilizing an enzyme reaction, the glucose concentration can be measuredin a short time and at a low voltage, any reductive substances containedin the glucose solution have little effect, resistance to exposure tolight or water is high, and the solubility of the reagent layer is high.

(Production of Glucose Sensor)

A glucose sensor with a first electrode, a second electrode, a reagentlayer and a channel formed on a substrate as shown in FIGS. 2 and 3, wasused in Examples 1 to 8. The first and second electrodes were formed onthe substrate by screen printing with a carbon paste.

Two glucose sensors were compared in Examples 1 to 6. One of these istermed glucose sensor 1 and the other comparative glucose sensor 1. Thedifference between these glucose sensors was in the formulation of theirreagent layers, as shown in Table 1 below. These reagent layers wereformed by applying spots of 1 μL of reagent composed of anoxidation-reduction enzyme and a potassium phosphate buffer on asubstrate, and then drying.

TABLE 1 Formulation of Reagent Layer Oxidation- Mediator reductionenzyme Buffer (pH 7) Glucose sensor 300 mM 5000 U/mL 50 mM 1 of present[Ru (III) (NH₃)₆]Cl₃ PQQGDH potassium invention phosphate 50 mMComparative 300 mM 5000 U/mL potassium glucose sensor 1 K₃[Fe (III)(CN)₆] PQQGDH phosphate

In Example 7, two glucose sensors 2 and 3 of the present inventioncomprising a different oxidation-reduction enzyme from that used inExamples 1 to 6 were used as indicated in Table 2 below. Other than theconstitution of the reagent layer, these were the same as the glucosesensors in Examples 1 to 6. αGDH and CyGDH were as discussed previously.

TABLE 2 Formulation of Reagent Layer Oxidation- Buffer Mediatorreduction enzyme (pH 7) Glucose sensor 2 300 mM 600 U/mL 250 mM ofpresent [Ru (III) (NH₃)₆]Cl₃ CyGHD potassium invention phosphate Glucosesensor 3 300 mM 600 U/mL 250 mM of present [Ru (III) (NH₃)₆]Cl₃ αGDHpotassium invention phosphate

A glucose sensor 4 of the present invention and a comparative glucosesensor 2 in which GOD was used as the oxidation-reduction enzyme asshown in Table 3 below were used in Example 8. Other than theconstitution of the reagent layer, these were the same as the glucosesensors in Examples 1 to 6.

TABLE 3 Formulation of Reagent Layer Oxidation- Mediator reductionenzyme Buffer (pH 7) Glucose sensor 300 mM 5000 U/mL 50 mM 4 of present[Ru (III) (NH₃)₆]Cl₃ GOD potassium invention phosphate 50 mM Comparative300 mM 5000 U/mL potassium glucose sensor 2 K₃[Fe (III) (CN)₆] GODphosphate

Example 1

In this example, the electrode response characteristics of glucosesensors were evaluated by examining CV waveforms. The CV waveform wasexamined by applying spots of glucose solution on the reagent layer ofthe glucose sensor, sweeping such that the sweep rate was 50 mV/sec andthe applied voltage was 0 mV→+800 mV→0 mV→−800 mV→0 mV→+800 mV, andmeasuring the response current during the sweep. The glucose solutionused here was a standard solution with a concentration of 200 mg/dL(prepared by dissolving glucose in physiological saline (0.9 wt %NaCl)). The amount of spot application of the glucose solution on thereagent layer was 1 μL. FIG. 6 shows the CV waveforms.

It can be seen from the CV waveforms in FIG. 6 that within a range inwhich the applied voltage was 0 mV→+800 mV on the second time, theresponse current value was at its maximum when the applied voltage wasapproximately 100 mV with the glucose sensor 1 of the present inventionin which [Ru(III)(NH₃)₆]Cl₃ was used as the mediator, whereas theresponse current value reached its maximum at slightly less than 300 mVwith the comparative glucose sensor 1 in which K₃[Fe(III)(CN)₆] wasused. The CV waveforms in FIG. 6 tell us that when [Ru(III)(NH₃)₆]Cl₃ isused as the mediator, if the applied voltage is set at 100 mV or higher,substantially all of the reductive compounds can be oxidized and madeoxidative, and similarly that when K₃[Fe(III)(CN)₆] is used, the appliedvoltage must be at least 300 mV. The applied voltage at which theresponse current value of each mediator reached its maximumsubstantially matched the standard oxidation-reduction potential foreach mediator.

Therefore it can be concluded that if a Ru complex with a low standardoxidation-reduction potential is used as the mediator, glucoseconcentration can be measured favorably even at a low applied voltage,and that in this case measurement at good precision is made possible bydecreasing the background current caused by other reductive substancespresent in the solution.

Example 2

In this example, it was examined whether glucose concentration can beaccurately measured at a low voltage (200 mV). To this end, the responsecurrent value was measured using four different standard solutions withglucose concentrations of 0 mg/dl, 200 mg/dL, 400 mg/dL, and 600 mg/dLand using the glucose sensor 1 of the present invention and thecomparative glucose sensor 1, at applied voltages of 500 mV and 200 mV.The response current value was measured 5 seconds after the spotapplication of 1 μL of standard solution to the reagent layer, with theapplication of voltage held steady between the first and secondelectrodes. These results are given in FIG. 7.

As can be seen from FIG. 7, when the applied voltage was 500 mV, theglucose sensor 1 of the present invention exhibited good linearity forthe group of plotted points, indicating that glucose sensor can bemeasured favorably even when the glucose concentration is high (400mg/dL or higher). In contrast, with the comparative glucose sensor 1,linearity was somewhat off when the glucose concentration was high (400mg/dL or higher), although the overall linearity was excellent.

Meanwhile, when the applied voltage was 200 mV, the linearity of theglucose sensor 1 of the present invention was off somewhat when theglucose concentration was high (400 mg/dL or higher), but the group ofplotted points did exhibit good linearity. The deviation in linearity at200 mV with the glucose sensor 1 of the present invention was stillsmaller than that of the comparative glucose sensor 1 at 500 mV.

Thus, it was confirmed that when a Ru complex was used as the mediator,glucose concentration could be measured favorably within a range ofglucose concentration of at least 0 to 600 mg/dl even at a low potential(applied voltage of 200 mV). Therefore, it can be concluded that the useof a Ru complex as the mediator makes it possible to drive theconcentration measuring apparatus at low voltage, which lowers powerconsumption and reduces running costs.

Example 3

In this example, it was examined how long it took to measure the glucoseconcentration favorably. To this end, 500 mV voltage application betweenthe first and second electrodes was commenced 0, 1, 2, or 10 secondsafter the spot application of 1 μL of whole blood with a glucoseconcentration of 400 mg/dL to the reagent layer, the response currentwas measured during sustained voltage application, and the change overtime was measured. These results are given in FIGS. 8 and 9.

It can be seen from FIG. 8 that with the glucose sensor 1 of the presentinvention, the individual measurement values obtained 3 seconds afterthe start of voltage application were the same, regardless of the timebefore voltage application (the time in a non-application state).Therefore, it can be concluded that the glucose sensor of the presentinvention gives stable measurement results as long as the applicationtime is at least 3 seconds, and, as seen in FIG. 9, that the applicationtime can be shorter than when an Fe complex is used as the mediator.Also, since there is no real advantage to excessively increasing thetime of the non-application state, and with the results of FIG. 8 inmind, it can be concluded that 10 seconds or less is an adequate time ina non-application state with the glucose sensor of the presentinvention, and that 10 to 15 seconds is sufficient for the time from theglucose solution supply until the response current is measured for thepurpose of computing the glucose concentration. Therefore, it can beseen from the results shown in FIGS. 8 and 9 that when a Ru complex isused as the mediator, the measurement does not take as long as when anFe complex is used.

Example 4

In this example, the effect of reductive concomitants (the effect ofbackground current) was examined. The response current value wasmeasured using four different types of whole blood (with componentsother than glucose adjusted to their average concentration in humanblood) with glucose concentrations of 0 mg/dL, 200 mg/dL, 400 mg/dL and600 mg/dL, and using the glucose sensor 1 of the present invention andthe comparative glucose sensor 1, at applied voltages of 500 mV and 250mV. There was a voltage non-application state lasting 10 seconds afterthe spot application of 1 μL of whole blood to the reagent layer, afterwhich the response current value was measured 5 seconds after the startof voltage application between the first and second electrodes. Theseresults are given in FIGS. 10 and 11.

As can be seen from FIGS. 10 and 11, the overall response current valuewas higher with the comparative glucose sensor 1 than with the glucosesensor 1 of the present invention. The reason for this seems to be thatthe comparative glucose sensor 1 is affected more by reductiveconcomitants in blood than is the glucose sensor 1 of the presentinvention, and that the background current from these substances raisesthe response current value.

It should be noted here that with the comparative glucose sensor 1, theresponse current is measured as a positive value even when the glucoseconcentration is 0 mg/dL. Here again, the effect of background currentcaused by reductive concomitants is suspected to be the reason for thehigher response current value with the comparative glucose sensor 1.

FIG. 12 is a bar graph of the response current value here when theglucose concentration was 0 mg/dL. It can be seen from this graph thatwith the comparative glucose sensor 1, a relatively large responsecurrent is measured even at a glucose concentration of 0 mg/dL, thereason for which is believed to be the greater effect of the reductiveconcomitants. In contrast, with the glucose sensor 1 of the presentinvention, the measured response current is small at a glucoseconcentration of 0 mg/dl, and it can be concluded that the effect of thereductive concomitants has been greatly reduced. Therefore, if a Rucomplex is used as the mediator, concentration can be computed with goodprecision even without correcting for the effect of other reductivesubstances.

Example 5

Exposure resistance was evaluated in this example. This resistance toexposure was evaluated by leaving the glucose sensor 1 of the presentinvention and the comparative glucose sensor 1, which had both beenproduced at about the same time, in a thermo-hygro-static room kept at arelative humidity of 50% and a temperature of 25° C., and then using astandard solution with a glucose concentration of 0 mg/dl to measure theresponse current value. The application of voltage of 500 mV between theelectrodes was started 10 seconds after the spot application of thestandard solution to the reagent layer, and the response current valuewas measured 5 seconds after the start of voltage application. Theglucose sensors were left 1 and 4 days inside the thermohygrostaticroom. The response current value was measured under the same conditionsboth for a glucose sensor that had yet to be left in thethermohygrostatic room (initial), and for a glucose sensor that had beensealed in a desiccator (interior volume of 0.2 L, initial relativehumidity setting of 50%, and temperature of 25° C.) containing 6 g ofmolecular sieve (desiccant) and then left for 4 days in athermohygrostatic room kept at a relative humidity of 50% and atemperature of 25° C. (sealed 4 days). These results are given in FIG.13.

As can be seen from FIG. 13, at all environmental settings, the glucosesensor 1 of the present invention had much smaller response current thanthe comparative glucose sensor 1, and the values thereof were about thesame at all environmental settings. It is therefore surmised that when aRu complex is used as the mediator, there is less degradation(reduction) of the reagent layer under the exposure environment, sostorage stability is superior and long-term degradation is less likelyto occur. Therefore, if a Ru complex is used as the mediator, there isno need to give much thought to the effect of exposure to moisture inthe storage of the glucose sensor. This means that when glucose sensorsare mass-produced and packaged on an industrial scale, there is no needto perform nitrogen replacement or other such treatment inside thepackaging, which facilitates manufacture and reduces costs.

Example 6

The solubility of the reagent layer was examined in this example. In theexamination of the solubility of the reagent layer, the response currentvalue was measured using four different types of standard solution withglucose concentrations of 0 mg/dL, 200 mg/dl, 400 mg/dL, and 600 mg/dLand using the glucose sensor 1 of the present invention and thecomparative glucose sensor 1, at an applied voltage of 500 mV. There wasa voltage non-application state lasting 10 seconds after the spotapplication of 1 μL of standard solution to the reagent layer, afterwhich the response current value was measured 5 seconds after the startof voltage application between the first and second electrodes. Theseresults are given in FIG. 14. FIG. 14 also shows the results for when aninorganic gel (used as a dispersant) was added in an amount of 1 weightpart per 100 weight parts Fe complex in the comparative glucose sensor1.

As can be seen from FIG. 14, the glucose sensor 1 of the presentinvention exhibited excellent linearity even at a high glucoseconcentration. This means that the reagent layer dissolves well within15 seconds of the supply of the standard solution, regardless of theglucose concentration.

Therefore, if a Ru complex is used as the mediator, the reagent layerwill have excellent solubility, with the entire reagent layer forming auniform reaction system, and as a result glucose concentrations can bemeasured in a short time and with good precision even with glucosesolutions having a relatively high glucose concentration, without havingto resort to the use of a dispersant or the like.

Example 7

In this example, the response current value was measured for the glucosesensors 2 and 3 of the present invention, which had a reagent layerformed as shown in Table 2 above, using four types of whole blood withdifferent glucose concentrations of 0 mg/dl, 200 mg/dL, 400 mg/dl, and600 mg/dL. These results are given in FIG. 15. FIG. 15 also shows theresults for when the response current value was measured under the sameconditions for the glucose sensor 1 of the present invention constitutedas in Examples 1 to 6.

As can be seen from FIG. 15, with glucose sensors 2 and 3 of the presentinvention, in which CyGDH or a GDH was used as the redox enzyme,concentration can be measured favorably from relatively lowconcentrations all the way up to high concentrations, under theapplication of a current of just 200 mV and only a short time of 5seconds after the start of application. FIG. 15 also tells us that thebackground current is small with these glucose sensors. Therefore, byusing a ruthenium complex as the mediator, the advantages described inExamples 1 to 6 can be achieved for various types of GDH.

Example 8

In this example, the response current value was measured in the samemanner as in Example 7 for a glucose sensor 4 of the present invention,in which a Ru complex was used as the mediator and GOD was used as theoxidation-reduction enzyme, and a comparative glucose sensor 2, in whichpotassium ferricyanide was used as the mediator and GOD was used as theoxidation-reduction enzyme. These results are given in FIG. 16.

As can be seen from FIG. 16, when GOD and a Ru complex are combined,just as with the glucose sensor 1 of the present invention used inExamples 1 to 6, linearity was excellent and background current wassmall even when the glucose concentration was relatively high. It cantherefore be concluded that the advantages described in Examples 1 to 6can also be achieved when GOD and a Ru complex are combined.

As described above, with the present invention, the concentration of atest target in a sample liquid can be accurately measured, whether theconcentration of the test target in the sample liquid is relatively lowor relatively high, the measurement will be affected less by thecoexistent reductive substances present in the sample liquid, andadequate solubility of the reagent layer can be ensured.

The invention claimed is:
 1. A method for measuring a concentration of atest target, the method comprising: constructing a reaction system bysupplying whole blood containing the test target to a reagent layercontaining an oxidation-reduction enzyme and an electron mediator;measuring the concentration of the test target by utilizing amperometry;wherein the test target is glucose, the oxidation-reduction enzyme isCyGDH, and a Ru compound ruthenium(III) complex is used as the electronmediator, the method further comprising: applying constant voltage tothe reagent layer; dissolving the electron mediator directly in thewhole blood containing the test target to form a solution; detecting thesolution by applying the voltage to the reagent layer for measurement;and computing the concentration of the test target on the basis of aresponse current value from the reagent layer; wherein the constantvoltage is no greater than 300 mV and selected from a range from astandard oxidation-reduction potential (versus a standard hydrogenelectrode) between an oxidant and a reductant of the Ru compoundelectron mediator through a standard oxidation-reduction potential(versus a standard hydrogen electrode) between ferrocyanide ions andferricyanide ions, wherein the concentration of the test target ismeasured at any point between 3 and 15 seconds after the whole bloodcontaining the test target is supplied to the reagent layer, and whereinthe system is configured to be without any interferent mitigatingelements.
 2. The method according to claim 1, wherein the Ru compound isoxidative Ru(III) complex expressed by the following chemical formula:[Ru(NH₃)₅X]^(n+) (where X in the formula is NH₃ or a halogen ion, and n+in the formula is the valence of the oxidative Ru(III) complex asdetermined by a type of X).
 3. A method for measuring a concentration ofa test target, the method comprising: constructing a reaction system bysupplying whole blood containing the test target directly to a reagentlayer containing an oxidation-reduction enzyme and an electron mediator;measuring the concentration of the test target by amperometry; whereinthe oxidation-reduction enzyme is selected from the group consisting ofglucose oxidase, CyGDH, cholesterol dehydrogenase, cholesterol oxidase,lactic acid dehydrogenase, lactic acid oxidase, ascorbic aciddehydrogenase, ascorbic acid oxidase, alcohol dehydrogenase, alcoholoxidase, fructose dehydrogenase, 3-hydroxybutyric acid dehydrogenase,pyruvic acid oxidase, NADH oxidase, uric acid oxidase (uricase), urease,and dihydrolipoamide dehydrogenase (diaphorase), and the electronmediator is a ruthenium(III) complex, the method further comprising:applying voltage to the reagent layer; dissolving the electron mediatordirectly in the whole blood containing the test target to form asolution; detecting the solution by applying the voltage to the reagentlayer for measurement; and computing the concentration of the testtarget on the basis of a response current value from the reagent layer;wherein the voltage is selected from a range of about 100 mV to 500 mV;wherein the concentration of the test target is measured at any pointbetween 3 and 15 seconds after the whole blood containing the testtarget is supplied to the reagent layer, and wherein the system isconfigured to be without any interferent mitigating elements.
 4. Themethod according to claim 3, wherein the voltage is selected from arange of about 100 mV to 350 mV.
 5. The method according to claim 3,wherein the voltage is selected from a range of about 100 mV to 300 mV.6. The method according to claim 3, wherein the test target is glucoseand the oxidation-reduction enzyme is glucose oxidase.
 7. A method formeasuring a concentration of glucose, the method comprising:constructing a reaction system by supplying whole blood containing theglucose to a reagent layer containing an oxidation-reduction enzyme andan electron mediator; measuring the concentration of the glucose byamperometry; wherein the oxidation-reduction enzyme is glucose oxidaseand the electron mediator is a ruthenium(III) complex the method furthercomprising: applying voltage to the reagent layer; dissolving theelectron mediator directly in the glucose to form a solution; detectingthe solution by applying the voltage to the reagent layer formeasurement; and computing the concentration of the glucose on the basisof a response current value from the reagent layer; wherein the voltageis selected from a range of about 100 mV to 350 mV; wherein theconcentration of the glucose is measured at any point between 3 and 15seconds after the whole blood containing the glucose is supplied to thereagent layer, and wherein the system is configured to be without anyinterferent mitigating elements.
 8. A concentration measuring apparatusand a measurement test instrument, wherein the measurement testinstrument comprises a reagent layer; a first electrode; and a secondelectrode, wherein the reagent layer contains an oxidation-reductionenzyme selected from the group consisting of glucose oxidase, CyGDH,cholesterol dehydrogenase, cholesterol oxidase, lactic aciddehydrogenase, lactic acid oxidase, ascorbic acid dehydrogenase,ascorbic acid oxidase, alcohol dehydrogenase, alcohol oxidase, fructosedehydrogenase, 3-hydroxybutyric acid dehydrogenase, pyruvic acidoxidase, NADH oxidase, uric acid oxidase (uricase), urease, anddihydrolipoamide dehydrogenase (diaphorase); and a ruthenium(III)complex, and wherein the concentration measuring apparatus comprises avoltage applier for applying voltage between the first and secondelectrodes; a current value measurer for measuring the response currentvalue when voltage selected from the range of about 100 mV to 500 mV isapplied between the first and second electrodes; a computer forcomputing the concentration of the test target on the basis of theresponse current value; and a controller for controlling the currentvalue measurement performed by the current value measurer, wherein thecontroller is constituted such that the response current value necessaryfor computation by the computer is measured by the current valuemeasurer at any point between 3 and 15 seconds after whole bloodcontaining the test target is supplied directly to the reagent layer toform a solution with the ruthenium(III) complex, and wherein theconcentration measuring apparatus and the measurement test instrumentare configured to be without any interferent mitigating elements.
 9. Theconcentration measuring apparatus and a measurement test instrumentaccording to claim 8, wherein the test target is glucose and theoxidation-reduction enzyme is glucose oxidase.
 10. The concentrationmeasuring apparatus and a measurement test instrument according to claim8, wherein the voltage is selected from a range of about 100 mV to 350mV.
 11. The concentration measuring apparatus and a measurement testinstrument according to claim 8, wherein the voltage is selected from arange of about 100 mV to 300 mV.
 12. The method according to claim 3,wherein the ruthenium(III) complex is provided as a micropowder in thereagent layer.
 13. The method according to claim 3, wherein theruthenium(III) complex is substantially uniformly dispersed in thereagent layer.
 14. The method according to claim 7, wherein theruthenium(III) complex is a micropowder in the reagent layer.
 15. Themethod according to claim 7, wherein the ruthenium(III) complex issubstantially uniformly dispersed in the reagent layer.
 16. Theconcentration measuring apparatus and a measurement test instrumentaccording to claim 8, wherein the ruthenium(III) complex is provided asa micropowder in the reagent layer.
 17. The concentration measuringapparatus and a measurement test instrument according to claim 8,wherein the ruthenium(III) complex is substantially uniformly dispersedin the reagent layer.
 18. The concentration measuring apparatus and ameasurement test instrument according to claim 8, wherein the first andsecond electrodes are formed by screen printing with a carbon paste. 19.The method according to claim 1, wherein the concentration of the testtarget is measured at any point between 3 and 5 seconds.
 20. The methodaccording to any one of claims 3, 4, and 5, wherein the voltage that isapplied to the reagent layer is constant voltage.
 21. The methodaccording to claim 4, wherein the voltage that is applied to the reagentlayer is constant voltage.
 22. The concentration measuring apparatus anda measurement test instrument according to any one of claims 8, 9, 10and 11, wherein the voltage that is applied is constant voltage.